The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and allow for response to tissue injury. We have identified a population of slowly cycling ISCs that is marked by telomerase (mTert) expression. mTert+ cells are rare single cells that distribute in a pattern along the crypt-villus axis similar to long-term label-retaining cells and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types and persist long term. Moreover, mTert-expressing cells appear to contribute to intestinal lineage development through both Lgr5-dependent and Lgr5- independent pathways. In order to investigate their role in intestinal cancer we stabilized ?-catenin within mTert+ cells, which resulted in their dramatic (1,500-fold) activation. Surprisingly, however, continuous canonical Wnt signaling in these cells did not give rise to intestinal adenoma formation, even after 6 months. This result stands in stark contrast to the effect seen following stabilization of ?-catenin within rapidly cycling Lgr5+ ISCs, which generate extensive adenomas within 4 weeks. Our studies support the concept that slowly cycling and rapidly cycling ISCs exhibit distinctly different responses to tumor initiating signals. We hypothesize that the lack of adenoma formation in mTert+ cells is due to a mechanism of enforced quiescence regulating these slowly cycling ISCs, which is lacking in rapidly cycling Lgr5+ ISCs. In order to better define the mechanisms leading to adenoma formation or repression, these studies will investigate the gene expression profiles of mTert- and Lgr5-expressing ISCs in the presence or absence of stabilized ?-catenin. To determine the role of microRNAs (miRNAs) in adenoma formation and repression we will perform global analysis of their expression patterns in the presence or absence of stabilized ?-catenin in mTert- and Lgr5-expressing ISCs. To determine whether miRNAs play a functional role in the repression of adenoma formation following stabilization of ?-catenin in mTert-expressing ISCs we will delete Dicer, the critical miRNA processing enzyme, within these cells to establish whether inactivation of this regulatory system results in adenoma formation. Similarly, we will delete Dicer in Lgr5-expressing ISCs to determine whether miRNAs have a functional role in the induction of adenoma formation. The results will generate new insight into the mechanisms underlying adenoma initiation in intestinal stem cells. PUBLIC HEALTH RELEVANCE: Our studies support the concept that slowly cycling and rapidly cycling intestinal stem cells exhibit distinctly different responses to tumor initiating signals. We hypothesize that the lack of adenoma formation in slowly cycling stem cells is due to a proliferation blocking mechanism, which is lacking in rapidly cycling stem cells. These experiments will isolate and characterize putative intestinal cancer stem cells generated by targeted stabilization of ?-catenin in both slowly cycling and rapidly cycling stem cells. Gene expression and microRNA analysis will identify key regulators of cancer stem cell formation and provide a basis to determine the critical changes leading to the development of cancer. Comparative analysis of these cells will provide unique insight into the mechanisms regulating the initial steps of cancer stem cell formation.